All cells have molecular mechanisms that ensure that they develop characteristic shapes with selective fitness advantages. For example, many bacteria assemble peptidoglycan cell walls in specific spatial and temporal patterns to take on rod-like shapes that promote motility and host invasion. The proteins that mediate bacterial cell-shape determination are largely known and there is substantial evidence that these peptidoglycan assembly and patterning proteins must be highly regulated. For example, different species modulate the same conserved shape determinants to establish different shapes, such as curved or helical rods. Even within a single cell, the cell shape determination machinery functions differently at different stages of the cell cycle and in different metabolic states. Despite the central importance of regulating cell shape, very little is known about the mechanisms that regulate peptidoglycan assembly proteins in different contexts. To understand the regulation of cell shape proteins, we are harnessing the unique features of Caulobacter crescentus, a gram-negative curved rod with a readily synchronized cell cycle and rich genetic and genomic toolkit. We propose three independent aims to dissect cell shape regulation. First, we will carefully quantitate the dynamics of the MreBCD, PBP1, PBP2, and RodAZ cell elongation proteins. We will determine how these dynamics are regulated by one another, by cell cycle progression, and by known cell cycle regulators. We will also image elongation protein dynamics in curvature mutants to understand how the elongation and curvature machineries inter-relate. Second, we will use our previously developed high-throughput screening tools and both in vivo and in vitro assays for MreB assembly to identify and characterize novel MreB regulators. These efforts will include characterizing the mechanisms of action of two MreB regulators that we recently identified, MbiA and AimB, as well as identifying novel proteins that modulate MreB. Third, we will exploit our recent discovery of CtpS as a bifunctional metabolic enzyme and cell shape regulator to define how shape and metabolism are co-regulated. Specifically, we will determine how metabolite levels influence both CtpS assembly and shape, how CtpS interacts with another curvature-determining polymer, crescentin, and the selective benefits of coupling shape to metabolic state. Our efforts will establish the first comprehensive understanding of how cell growth is regulated in different states and how it is coupled to other cellular processes such as division and metabolism. These studies will provide a roadmap for cell shape studies in other systems. Furthermore, the shape-determining bacterial cell wall represents one of the most common targets for antibiotic drugs. Thus, our work will aid in the development of future generations of antibiotics to replenish the therapeutic arsenal that is being rapidly depleted by the rise of resistance to existing drugs.